Human Menkes and Wilsons diseases are caused by homeostatic defects of a metal ion, copper (Cu2+ and Cu+). Copper ion is a dynamic biological nature, essential yet toxic. The two diseases represent the two extremes in copper ion homeostasis: copper ions deficiency and excess. Copper ion deficiency caused loss of function of copper ion-dependent enzymes leads to Menkes disease. Copper ion excess mediated toxicity is Wilson's disease. Since the copper ion is an essential nutrient yet a potent toxin, cells must possess a dynamic homeostasis to maintain a proper cellular level of copper ions. What is the molecular mechanism of dynamic copper ion homeostasis? This is still a fundamental question to be addressed. The goal of this proposal is, using yeast as a model system, to study the dynamic regulation in copper ion homeostasis. Yeast cells carry out similar biochemical reactions to those required in humans. Furthermore, copper ion homeostatic mechanisms are largely conserved among yeast, human and other divergent species. Mac1p, a putative metal ion sensing yeast protein, functions as a key regulatory factor in high affinity copper ion uptake and transport under physiological conditions. Recent studies have demonstrated that Mac1p responds to toxic levels of copper ions through an uncharacterized mechanism of regulated protein degradation. Mac1p degradation is a new mechanism in copper ion homeostasis and independent of its regulatory function in copper ion transporter gene CTR1 and CTR3 expression. The differential functions of Mac1p under physiological and toxic copper ion conditions reflect the dynamic biological nature of copper ions. Therefore, understanding Mac1p functions will be instrumental to determine molecular mechanism of dynamic copper ion homeostasis. In this proposal, a combination of genetic, molecular biology, chemical and biochemical methods are designed to determine the precise molecular mechanisms of Mac1p functions. These approaches will be applied to address the following questions: (1) how does Mac1p respond differentially to physiological and toxic levels of copper ions? (2) how does Mac1p sense copper ion and its concentration changes? (3) What is the molecular mechanism and importance of regulated protein degradation in copper ion homeostasis? The outcomes of these proposed research will make fundamental contributions to the understanding of dynamic copper ion homeostasis.